Androgen and Progesterone Binding Components in Cytosol Prepared from Cultures Enriched in Sertoli Cells from Immature Rat Testes1

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Abstract
Sephadex G-25 chromatography has been used to examine cytosol prepared from 5-6-day-old cultures of immature rat Sertoli cells for the presence of macromolecular androgen and progesterone binding components. High affinity, slowly dissociable, androgen binding activity was detected that exhibited first-order half-times of dissociation (t½) of 3.2 ± 1.0 h for [3H]-testosterone and 3.5 ± 1.3 h for [3H]-5α-dihydrotestosterone. These dissociation rates clearly distinguish this androgen binding component from Androgen Binding Protein (ABP), which has a t½ of ∼5 min. Specific binding of [3H]-testosterone to the non-ABP binding moiety was completely inhibited by a 100-fold molar excess of unlabeled testosterone, 5α-DHT, Δ6-testosterone, and progesterone. Specific binding of [3H]-progesterone was also observed. Approximately 20%-40% of the specific binding persisted 30 min after the addition of 100-fold molar excess of unlabeled progesterone to samples that had been equilibrated with [3H]-progesterone. Analysis of the dissociation of [3H]-progesterone bound to macromolecules with time revealed the presence of at least two binding components. One component showed a rapid rate of dissociation (t½ ∼ 13 min), and the other component showed a slow rate of dissociation (t½>5 h). Specific binding of [3H]-progesterone to the slowly and rapidly dissociating components showed similar but not identical sequences of binding specificity (slow component: P>5α-DHT>T = Δ6-T>R5020>MPA = E = F; rapid component: P>5α-DHT>R5020>T = 6Δ-T>MPA = E = F). Studies on the metabolism of [3H]-5α-DHT, [3H]-testosterone and [3H]-progesterone by Sertoli cell cytosol (3 h, 0°C-2°C) revealed that: 1) ∼50% of the [3H]-5α-DHT was converted to [3H]-5α-androstan-3α, 17α-diol, and 2) little (3H]-testosterone or (3H]-progesterone occurred. When the radioactivity specifically bound to macromolecular components in cytosol was examined, we determined that: 1) in incubations with [3H]-5α-DHT, 90% of the bound label was [3H]-5α-DHT; 2) in incubations with [3H]-testosterone, greater than 94% of the bound label was [3H]-testosterone; and 3) in incubations with [3H]-progesterone, greater than 98% of the bound label was [3H]-progesterone.